Preclinical Approaches to the Protection of Ovarian Function

9.1

Introduction

Recent advances in our understanding of the mechanisms underlying the impact of cytotoxic drugs on the ovary have opened up new directions for the protection of ovarian function from chemotherapy-induced damage. Studies are providing greater detail as to the pathways and factors triggered within the different cell types of the ovary by each drug class. As we gain increased knowledge of the specifics of these pathways, we reveal new targets for protective agents to reduce or prevent ovarian damage.

Most preclinical research on protective agents has concentrated on drug groups known clinically to have severe effects on follicle reserve, such as alkylating agents (cyclophosphamide, busulfan and dacarbazine), platinum complexes (cisplatin, carboplatin) and taxanes (paclitaxel). A large number of studies have also examined the anthracyclin antibiotic doxorubicin (DXR), which has been shown to affect the follicle reserve experimentally but which is currently considered of low clinical risk for premature ovarian insufficiency (POI). Alkylating agents and platinum complexes work in a similar fashion, creating DNA crosslinks, which, in turn, cause DNA breaks, ultimately triggering apoptosis. The taxanes are microtubule-stabilizing agents, as distinct from DNA-damaging drugs, but it has been demonstrated that paclitaxel acts via Bax to induce apoptosis. DXR is an intercalating agent that blocks DNA replication and causes double-stranded (ds)DNA breaks; it induces apoptosis primarily in the stroma and granulosa cells of growing follicles. Within the oocyte, anthracyclin agents such as DXR have been shown to induce chromosomal fragmentation as well as fragmentation of the cytoplasm into apoptotic bodies.

9.2

Ovarian Protection by Affecting Apoptotic Pathways

The most extensively researched pathway in the context of chemotherapy- and radiotherapy-induced ovarian injury is the apoptotic pathway leading to cell death in response to DNA damage. Cytotoxic drug-induced apoptosis has been most clearly demonstrated in growing follicles ( Figure 9.1B ), and has been shown to originate in the proliferating granulosa cells. Within the mature oocyte, apoptosis is mediated by several molecules including ceramide, Bax, and the caspases. Ceramide has been identified as an initiator of DXR-induced apoptosis in oocytes, and Bax, a protein produced by the Bcl2-gene family, plays an essential role in DXR-initiated apoptosis in mature mouse oocytes. Caspases, members of the CASP protease family, have been universally shown to play a pivotal role as cell death effector molecules, and are central in the apoptotic pathway of mature oocytes. Caspases-2, -12 and -3 specifically, have been shown to play a role in apoptosis of mature oocytes. Mouse oocytes pretreated with a caspase inhibitor as well as mature oocytes derived from mice that lack expression of caspase-2 show a marked resistance to DXR-induced apoptosis.

Investigation of apoptotic pathways within the dormant primordial follicle have revealed that p63, a homologue of anti-oncogene p53, found in the nucleus of oocytes, and specifically the p63 isoform TAp63, is a key mediator of the DNA damage apoptosis pathway in the response of primordial follicle oocytes to DNA injury. The p63 pathway is upregulated when oocytes are exposed to external triggers of DNA damage such as radiation and chemotherapy drugs such as cisplatin, and loss of p63 in mouse oocytes results in resistance to the apoptotic effects of radiation and cisplatin. Tap63 activates apoptosis via proteins BAX (Bcl-2-associated X protein) and BAK (Bcl-2 homologous antagonist killer). The activation of apoptosis is mediated by TAp73, either directly or through the activation of p53 upregulated modulator of apoptosis (PUMA) and phorbol-12-myristate-13-acetate-induced protein 1 (NOXA). Oocyte-specific deletion of PUMA and/or both PUMA and NOXA in mice prevents γ-irradiation-induced apoptosis and can produce healthy offspring, indicating that the protected oocytes are capable of DNA repair and subsequent normal function. Figure 9.1 schematically summarizes the key factors in the relevant apoptotic pathways.

Each of these factors in the apoptotic cascade represents a potential target for blocking the effects of cytotoxic treatments on the ovary. One important concern with developing any agent that interferes with the apoptotic pathway as a potential protectant is that, since induction of apoptosis is a key anticancer action of many of the chemotherapy drug classes, interference with the apoptotic pathway may inhibit the anticancer effects of these drugs. A further concern is that blocking apoptosis in DNA-damaged oocytes could allow survival of germ cells that are beyond the repair capabilities of the DNA repair molecules. Fertilization of these genetically compromised oocytes could lead to an increased risk of miscarriage, fetal death or malformation.